The adaptive response (AR) is a phenomenon whereby the harmful effects of high dose ionizingradiation or other genotoxic agents can be mitigated by prior exposure to a low dose of the same or similar genotoxic stress. The adapted cells show an increased survival, less chromosomalaberration and decreased mutagenesis termed the adaptive response. It is not clear which biologicpathways are involved in the AR, speculation centers on cell cycle controls, signal transduction,and DNA repair mechanisms. DNA repair mechanisms, once thought to be constitutive, havenow been proven to be inducible. Wilson, Mitra, and others have shown that genes and geneproducts involved in base excision repair are induced after low doses of certain forms of DNAdamaging agents. We are working on the hypothesis that induced DNA repair, both base excisionand nucleotide excision repair are equally important as underlying mechanisms of AR. Thecomponents of the proximal limb of the p53 DNA damage response pathway likely are critical inthe initiation and maintenance of the adaptive response. We believe that the most interestinghypotheses to construct center on understanding the role of p53 damage response pathways in theadaptive response. Specifically, we hypothesize that DNA repair induced by lowdoses of ionizing radiation occurs through induction or activation of p53 related genes. There isevidence that PARP and ATM are required for AR. However, the role of DNA-PK in concertwith these two components and possible c-ABL is unclear. It is also not discerned how otherp53 related genes such as interferon regulatory factors 1 and 2 (IRF1,2), GADD45, p21 areinvolved. GADD45 known to be induced by low-moderate doses of ionizing radiation and IRF1 a tumor suppresser protein and transcription factor known to regulate responses to DNAdamage by interacting with p53 and p21 have yet to be examined in the AR pathway. Wespeculate that these genes are critical elements in the AR because they are essential in the DNAdamage recognition mechanisms, and cell cycle check points and may activate or inducenucleotide excision and/or base excision repair. Although Human AP Endonuclease 1 has been implicated as a possible gene involved in the induction of base excision repair after low doses of oxidative damage, it is not known if nucleotide excision repair related genes may play a comparable role.Our work to date has focused on evaluating the role of DNA-PK in AR. Using SCID mousemodels with different mutations in DNA-PK, we are evaluating AR in terms of biologicalendpoints that include apoptosis, cell survival, and persistence of DNA damage measured by theSingle Cell Gel Electrophoresis (COMET Assay) after low doses of gamma irradiation. Other mutant cell lines with defects in p53 related genes and DNA repair related genes will be used as well to dissect the pathways involved. We chose to examine the role of DNA-PKcs in AR using a SCID mouse model, because DNA-PK is one of the initial molecules to recognize DNA damage and because it has been implicated in different aspects of DNA repair, a mechanism believed to play a role in AR. Our data indicate that DNA-PKcs may not be essential for the induction of AR in the SCID mouse model when evaluated in terms of apoptosis and comet assay as end points. It is interesting to note that even though, DNA-PK is an important component of the DNA damage and repair mechanisms, it may not be involved in the augmentation of DNA repair that occurs following low doses of radiation. By examining these endpoints, we clearly demonstrate that SCID cells, deficient in DNA- PKcs, show AR to the same extent seen in the wild type cells. Therefore, we conclude that DNA-PKcs is not essential for AR. It is known that these SCID cells have defects in double strand break and nucleotide excision repair as well. There are no reports on altered base excision repair pathway (BER) in these cell lines. Since we believe that enhanced DNA repair is a mechanism of the adaptive response , we hypothesize that base excision repair is competent in these SCID cells. If SCID cells show AR, then the DNA repair in these cells are accentuated via BER pathway. In order to document enhanced BER in SCID cells, we examined APE-1 protein expression in the adapted and non-adapted cells. Ramana et al., have shown an up regulation of APE-1 gene following low levels of oxidative stress and that APE-1 was used as an indicator of BER. While the low dose priming irradiation did not induce APE-1 proteins, the adapted cells showed an exaggerated and more sustained expression of APE-1 than the non-adapted ones. The induction of APE-1 most probably occurred at the level of transcription, because the increased APE-1 transcripts were also seen in adapted cells on northern blot analysis. This shows that BER is unregulated in the process of AR, in SCID cells. The control NF22 cells also showed similar response of APE-1 expression indicating that BER is equally accentuated in normal cells as well. Therefore, it appears that AR results from enhanced DNA repair, with major contribution from enhanced BER pathway.
